U.S. patent application number 10/071488 was filed with the patent office on 2002-07-18 for highly selective butyrylcholinesterase inhibitors for the treatment and diagnosis of alzheimer's disease and dementias.
Invention is credited to Brossi, Arnold, Greig, Nigel H., Hausman, Marvin, Soncrant, Timothy T., Yu, Qian-Sheng.
Application Number | 20020094999 10/071488 |
Document ID | / |
Family ID | 21975382 |
Filed Date | 2002-07-18 |
United States Patent
Application |
20020094999 |
Kind Code |
A1 |
Greig, Nigel H. ; et
al. |
July 18, 2002 |
Highly selective butyrylcholinesterase inhibitors for the treatment
and diagnosis of Alzheimer's disease and dementias
Abstract
The present disclosure relates to the discovery that highly
selective butyrylcholinesterase inhibitors can prevent or treat
cognitive impairments associated with aging or Alzheimer's disease.
A preferred butyrylcholinesterase inhibitor is cymserine.
Inventors: |
Greig, Nigel H.; (Phoenix,
MD) ; Yu, Qian-Sheng; (Baltimore, MD) ;
Brossi, Arnold; (Bethesda, MD) ; Soncrant, Timothy
T.; (Silver Spring, MD) ; Hausman, Marvin;
(Stevenson, WA) |
Correspondence
Address: |
Intellectual Property Docket Administrator
Gibbons, Del Deo, Dolan, Griffinger & Vecchione
One Riverfront Plaza
Newark
NJ
07102-5497
US
|
Family ID: |
21975382 |
Appl. No.: |
10/071488 |
Filed: |
February 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10071488 |
Feb 7, 2002 |
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09254494 |
Jun 17, 1999 |
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09254494 |
Jun 17, 1999 |
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PCT/US98/14063 |
Jul 9, 1998 |
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60052087 |
Jul 9, 1997 |
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Current U.S.
Class: |
514/411 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 25/00 20180101; A61K 31/40 20130101; C07D 491/048 20130101;
C07D 487/04 20130101; C07D 495/14 20130101; A61K 31/407
20130101 |
Class at
Publication: |
514/411 |
International
Class: |
A61K 031/407 |
Claims
What is claimed is:
1. A method for preventing or treating cognitive impairments
associated with aging or Alzheimer's disease which comprises
treating a patient at risk for or having said cognitive impairment
with an effective amount of a highly selective
butyrylcholinesterase inhibitor.
2. The method of claim 1 wherein said butyrylcholinesterase
inhibitor has a selectivity ratio of butyrylcholinesterase
inhibition to acetylcholinesterase inhibition of greater than about
15 to 1.
3. The method of claim 2 wherein said butyrylcholinesterase
inhibitor is selected from the group consisting of cymserine,
N'-norcymserine, N'-benzylnorphysostigmine, N'-benzylnorcymserine,
N'-phenethylnorphysosti- gmine, N'-phenethylnorcymserine,
N'-allylnorphysostigmone, thiaphysovenine, thiacymserine,
cymsvenine, N.sup.8-benzylnorcymserine, N.sup.8-norcymserine, N',
N.sup.8-bisnorcymserine, N.sup.1,
N.sup.8-Bisbenzylnorphysostigmine, N.sup.1,
N.sup.8-bisbenzylnorphmserine- , N.sup.1,
N.sup.8-bisbenzynorcymserine, and their pharmaceutically acceptable
salts.
4. The method of claim 3 wherein said butyrylcholinesterase
inhibitor is cymserine.
5. The method of claim 3 wherein said butyrylcholinesterase
inhibitor is N.sup.1, N.sup.8-bisnorcymserine
6. A compound having the formula of N.sup.8-benzylnorcymserine.
7. A compound having the formula of N.sup.8-horcymserine.
8. A compound having the formula of N.sup.2,
N.sup.8-bisnorcymserine.
9. A compound having the formula of N.sup.1,
N.sup.8-bisbenzylnorphysostig- mine.
10. A compound having the formula of N.sup.1,
N.sup.8-bisbenzylnorphenseri- ne.
11. A compound having the formula of N.sup.1,
N.sup.8-bisbenzylnorcymserin- e.
12. A method for detecting the presence of lesions or pathologic
states associated with Alzheimer's disease which method comprises
treating brain tissue with a reagent comprising a highly selective
butyrylcholinesterase inhibitor bound to a detectable label whereby
said reagent selectively binds to said lesions or pathologic states
which are detected by detecting the associated label.
13. The method of claim 12 wherein said reagent is a
pharmaceutically acceptable formulation which is administered in
vivo to a patient and passes into the brain tissue, said label
being detectable by suitable brain scan equipment and the presence
of said lesions or pathologic states are detected by carrying out a
brain scan on said patient.
14. The method of claim 13 wherein said butyrylcholinesterase
inhibitor is cymserine which contains a carbon.sup.11 label.
15. The method of claim 12 wherein said tissue is fixed to a slide
and said label is a fluorescent label.
16. A method for lowering secretion and synthesis of beta amyloid
precursor protein from cell lines or tissues or neuronal origin
which method comprises administering to said cell lines or tissue
of neuronal origin an effective amount of a highly selective
butyrylcholinesterase inhibitor.
17. The method of claim 16 wherein said butyrylcholinesterase
inhibitor is selected from the group consisting of cymserine,
N'-norcymserine, N'-benzylnorphysostigmine, N'-benzylnorcymserine,
N'-phenethylnorphysosti- gmine, N'-phenethylnorcymserine,
N'-allylnorphysostigmone, thiaphysovenine, thiacymserine,
cymsvenine, N.sup.8-benzylnortolserine, N.sup.8-benzylnorcymserine,
N.sup.8 nortolserine, N.sup.8-norcymserine, N,
N.sup.8-bisnorphysostigmine, N, N.sup.8-bisnorphenserine,
N',N.sup.8-bisnorcy, serine and their pharmaceutically acceptable
salts.
18. The method of claim 16 wherein said butyrylcholinesterase
inhibitor is cymserine.
Description
BACKGROUND OF THE INVENTION
[0001] Defects in the cholinergic system have been suggested to
underlie cognitive impairments associated with normal aging and
Alzheimer's disease (Bartus et al., Science 217:408-417 (1982);
Fisher et al., Neurobiol. Aging 13:9-23 (1992)). Much research has
focused on the development of cholinomemetic replacement therapy as
a potential treatment of these impairments. Among them,
cholinesterase inhibitors, such as physostigmine (Phy) and
tetrahydroaminoacridine (ThA) have been investigated for
memory-enhancing effects in both animals (Rupniak et al.,
Neurobiol. Aging 11:09-613; 1990); Murray et al.,
Psychopharmacology 105:134-136(1991) and human patients (Mohs et
al., J. Am. Geriatr. Soc. 3:749-757 (1985); Summers et al., N.
Engl. J. Med. 315:1241-1245(1986)).
[0002] Other agents have been proposed as selective inhibitors of
acetylcholinesterase (AChE). Thus heptyl-physostigmine (Heptyl-Phy)
was described as having greater lipophilicity, longer inhibitory
action on cholinesterase and more persistent increases in
acetylcholine in brain with less toxicity than the parent compound
(Brufani et al., Pharmacol. Biochem. Behav. 26:625-629 (1987)).
There is concern, however, as to whether the therapeutic window of
heptyl-Phy is wide enough for clinical use. Phenserine
((-)-N-phenylcarbamoyl eseroline) has been identified as a
superior, selective AChE inhibitor and thus suited as an agent for
the therapy for cognitive impairments associated with aging and
Alzheimer's disease. (U.S. Pat. No. 5,409,948, issued Apr. 25,
1995). In U.S. Pat. No. 5,171,750 issued Dec. 15, 1992, a series of
substituted phenserines are disclosed which are indicated to be
either selective inhibitors of AChE or butyrylcholinesterase
(BChE). The cumylcarbamate (4'-isopropylphenylcarbamate) derivative
of (-)-physovenol was noted to have a reverse enzyme specificity,
i.e., it inhibited BChE selectively over AChE. The patent indicates
that the compounds of the invention are useful "for treating
cholinergic disorders such as glaucoma, Myasthenia Gravis,
Alzheimer's disease and as an antidote against poisoning with
organo phosphates." There is no indication as to which type of
inhibitor would be used to treat the specified disorders, however,
there is a further disclosure to the effect that AChE, which is
found in red blood cells, in the brain and in nerve tissues, seems
to be more specific an enzyme known to hydrolyze acetylcholine
(ACh) in vivo than does BChE which is found in serum, pancreas and
the liver. The marked cholinergic loss in AD is accompanied by
dramatic reductions in the enzymes cholineacetyl transferase,
involved in the synthsis of the cholinergic neurotransmitter
acetylcholine, Ach, and of AChE, that ends the action of Ach
(Perry, et al. Brit. Med. J., 2 6150: 1457-1459, 1978; Whitehouse,
et al. Science 215; 1237-1239, 1982.
[0003] U.S. Pat. No. 5,378,723, issued Jan. 3, 1995 describes a
series of thiaphysovenol carbamic acid derivatives which are
indicated to exhibit high potency in the inhibition of AChE or
BChE. The compounds of that invention were indicated, as in the
case of U.S. Pat. No. 5,171,750 above, to be useful in treating
disorders such glaucoma, Myasthenia Gravis, Alzheimer's disease and
poisoning with organo phosphates. As above, no specific indication
is given as to which type of inhibitors would be used in which
specified disorder.
[0004] Geula and Mesulam in a paper entitled "Cholinesterases and
the Pathology of Alzheimer's Disease", Alzheimer's Disease and
Associated Disorders, Vol. 9, Suppl. 2, pp 23-28 (1995) make the
following observations in summary: "Alzheimer's Disease (AD) is
accompanied by a marked loss of acetylcholinesterase (AChE)
activity associated with cortical cholinergic axons and
cholinoceptive neurons. Simultaneous with this loss, cholinesterase
(ChE) activity emerges in AD cortex in the form of AChE and BChE
activity associated with plaques, tangles, and amyloid angiopathy.
Our observations have shown that the ChE's associated with the
pathological lesions of AD (ADChEs) possess different enzymatic
properties and quite possibly are of a different source as compared
with the ChEs associated with normal neurons and axons. The ADChEs
most likely have noncholinergic functions involved in the
pathogenesis of AD." In a further section the authors at p.26
state: "These observations indicate that glia are a likely source
of the ChE, and particularly the BChE, associated with the
pathological lesions of AD. They also suggest that a high ratio of
BChE to AChE positive glia may play a permissible or causative role
in the neuropathology of this disease. It is possible that other
pools of ChE exist with enzymatic properties similar or identical
to those of AD ChEs. This possibility remains unexplored."
[0005] Workers in the art have indicated that BChE is found in
significantly higher quantities in AD plaques than in plaques from
age-matched non-demented brains. Moreover, BChE was found to alter
the aggregation of beta amyloid peptide (A.beta.). It has been
hypothesized that since AChE is inhibited by high concentrations of
acetylcholine (ACh), while BChE remains unaffected, it may well be
that BChE may play an important role in the in vivo regulation of
synaptic concentrations of ACh in the brain of AD patients. BChE
inhibitor, instilled into the brain has produced a significant
increase in the level of extracellular Ach. (Giacobini, et al.,
Proc. Soc. Neurosci., 22; 203, 1996.)
[0006] It has also been found that in 3 AD specimens plaques, which
were of the compact or neuritic type, were almost always associated
with intense BChE activity. It was concluded that BChE activity
appears at the intermediate stage of plaque formation and that it
may therefore constitute one of the factors involved in the
transformation of an initially benign A.beta. deposit into a
compact neuritic form associated with neural degeneration and
dementia.
SUMMARY OF THE INVENTION
[0007] It has been unexpectedly discovered and thus forms the basis
of the present invention that highly selective BChE inhibitors can
be utilized by systemic administration to prevent or treat
cognitive impairments associated with aging or Alzheimer's disease
in a host. Since BChE activity has previously been identified to
reside primarily in peripheral organs, such as the pancreas, liver
and serum or in the circulation and its inhibition was associated
with side effects observed in first generation Alzheimer's disease
therapy (Liston et al., Proc. Soc. Neurosci., 20: 608, 1994.) Soreq
& Zachnt, Human Cholinestrase and ?, Academic Press, New York,
pp. 21-29, 1993) ref), the use of highly selective BChE inhibitors
in the treatment or prevention of cognitive impairments associated
with aging or Alzheimer's disease was not suggested by the art. A
further factor that pointed away from the possible use of highly
selective BChE inhibitors in treating cognitive diseases of the
brain and CNS was the expected distribution pattern of such agents.
Data available in the art suggest that such compounds would be
preferentially bound to peripheral organs where the major part of
their substrate activity resides. It was, therefore, not expected
that clinically useful concentrations of highly selective BChE
inhibitors administered systematically to a patient would pass
through the blood brain barrier and be available in the brain as
(i) such compounds would be expected to be bound to systemic enzyme
before reaching the brain, restricting its access, and (ii) most
BChE inhibitors known in the art do not readily enter the brain.
Indeed, until the present, inhibitors of BChE have been largely
utilized as pesticides in agriculture. (Soreq and Zachut, Human
Cholinesterases and Anticholinesterases, Academic Press, N.Y., pp.
21-29, 1993).
[0008] The term "highly selective" as used herein is meant to
include those BChE inhibitors whose ratio of IC (50) values against
human plasma BChE compared to their IC (50) values against human
erythrocyte AChE were greater than about 15 to 1.
[0009] The IC (50) values can be determined for such inhibitors
using methods well known in the art. In such assay the
pharmacological activity of each compound, as an IC (50), defined
as the concentration, in nanomoles, required to inhibit 50% of the
enzyme activity of AChE and BChE, is determined separately. For
determination of IC (50) values, the enzyme activity of each
concentration was expressed as a percent of that determined in the
absence of each compound. This then was transformed into a logit
format, where logit=In (% activity/[100-% activity]), and was
plotted as a function of the log concentration of the compound. IC
(50) values (i.e., logit=In (50/[100-5O]=0) were determined only
from correlation coefficients (r.sup.2) of less than -0.985 and
when more than 50% inhibition was achieved from duplicate samples.
The selectivity ratio is then determined by comparing the IC (50)
values obtained for each compound with AChE to that for BChE.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIGS. 1A, 1B and 1C represent a table of chemical structures
of compounds tested for activity and includes compounds with
desirable selectivity for BChE in accordance with the present
invention.
[0011] FIG. 2 is an immunoblot of an assay to determine the effect
of indicated compounds on the in vitro secretion of .beta.APP.
[0012] FIG. 3 is a graph demonstrating that cymserine reduces CSF
.beta.APP.gamma.. levels in rats.
[0013] FIG. 4 is a chart showing the blood/brain barrier
distribution in a rat over time after administration of 1 mg/kg I.V
cymserine.
[0014] FIG. 5 is a graph illustrating that cymserine improves
cognitive performance in rats.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The highly selective BChE inhibitors useful in the method of
the present invention are those compounds in Table 1 which are
indicated to have a selectivity for BChE of 15 or greater, while
their structures are provided in FIGS. 1A, 1B and 1C (along with
their selectivity ratio) which for compound of the invention again
is a selectivity for BChE of 15 or greater. For structural
comparison purposes, other related compounds are also shown in
Table 1 and FIGS. 1A, 1B, 1C which do not exhibit the desired
selectivity for BChE.
[0016] Among the compounds listed in Table 1 and FIG. 1 are certain
novel compounds which inhibit butyrylcholinesterase. The novel
compounds of the invention are (number in parenthesis corresponds
to number of compound in Table 1):
N.sup.8-benzylnorcymserine.sup.(33); N.sup.8-norcymserine.sup.(-
37); N.sup.1, N.sup.8-bisnorcymserine (41); N.sup.1,
N.sup.8-bisbenzylnorphysostigmine (42); N.sup.1,
N.sup.8-bisbenzylnorphen- serine (43); and N.sup.1,
N.sup.8-bisbenzylnorcymserine (45). These novel compounds were
synthesized as follows:
[0017]
(-)-(3aS)-8-Benzyl-1,3a-dimethyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-
-b]indol-5-yl-N-4'-isopropylphenylcarbamate
(N.sup.8-Benzylnorcymserine; compound 33).
(-)-(3aS)-8-Benzyl-1,3a-dimethyl-1,2,3,3a,8,8a-hexahydropyr- rolo
[2,3-b]indol-5-ol.sup.1 (33 mg, 0.112 mmol) was dissolved in ether
(2 mL), and Na (1 mg) was added. The mixture was stirred at r.t.
for 1 min, then 4-isopropylphenylisocyanate (18.1 mg, 0.112 mmol)
was added. The mixture was stirred at rt for 5 min. After the
removal of solvent, the residue was chromatographed
(CH.sub.2Cl.sub.2/MeOH=20/l) to give 33 (40 mg, 80.0%) as a foam:
[.alpha.].sub.D -60.0.degree. (c=0.2, CHCl.sub.3); .sup.1H NMR
(CDCl.sub.3) .delta.7.40-7.08 (m, 9H, Ar--H), 6.80 (d, J=2.2 Hz,
1H, C4-H), 6.70 (dd, J=2.2, 8.5 Hz, 1H, C6-H), 6.15 (d, J=8.5 Hz,
1H, C7-H), 4.45 and 4.35 (AB, J=16.6 Hz, 2H, Ph--CH.sub.2), 4.25
(s, 1H, C8a-H), 2.80 (m, 1H, Ph--CH<), 2.68 (m, 2H, C1-H.sub.2),
2.32 (s, 3H,N1-CH.sub.3), 1.90 (m, 2H,C2-H.sub.2), 1.35 (s, 3H,
C3a-CH.sub.3), 1.15 (d, J=7.0 Hz, 6H,>CMe.sub.2); EI-MS m/z
(relative intensity): 294 (MH+--ArNHCO, 65), 280 (2.2), 265 (3.6),
237 (75), 207(58), 160 (34), 91 (100). HR-MS m/z Calcd for
C.sub.29H.sub.33N.sub.3O.sub.2: 455.2573; found: 455.2569.
[0018]
(-)-(3aS)-1,3a-Dimethyl-1,2,3,3a,8,8a-hexhydropyrrolo[2,3-b]indol-5-
-yl-N-4'-isopropylphenylcarbamate (N.sup.8-Norcymerserine; compound
37). Compound 33 (22 mg, 0.048 mmol) was dissolved in a mixture of
MeOH (1 mL), H.sub.2O (1 mL) and TFA (0.5 mL). Palladium hydroxide
on carbon (5 mg) was added. The reaction mixture was stirred under
hydrogen at atmospheric pressure at r.t. for 1 h, and then the
catalyst was filtered. The filtrate was evaporated in vacuo to give
a residue which was dissolved in H.sub.2O, basified by
Na.sub.2CO.sub.3, extracted with ether, then dried over
Na.sub.2SO.sub.4. After removal of solvent, the residue was
chromatographed on a preparative TLC (silica gel)
(CH.sub.2CI.sub.2=10/1) to give product 37 (12 mg, 65.7%) as gum:
[.alpha.]D-73.8.degree.(c=0.2, CHCl.sub.3); .sup.1HNMR (CDCl.sub.3)
.delta.67.30 (d, J=8.5 Hz, 2H, C2'-H and C6'-H), 7.10 (d, J=8.5 Hz,
2H, C3'-H and C5'-H), 6.80-6.70 (m, 2H, C4-H and C6-H), 6.50 (d,
J=8.5 Hz, C7-H), 4.65 (s, 1H, C8a-H), 2. 85 (m, 2H, C2-H.sub.2),
2.64 (m, 1H, --HC<), 2.48 (S, 3H, N1-CH.sub.3), 2.00-1.90 (m,
2H, C3-H.sub.2), 1.42 (s, 3H, C3a-CH.sub.3), 1.20 (d, J=7.0 Hz, 6H,
>CMe.sub.2); EI-MS m/z (relative intensity): 204
(MH.sup.+--ArNHCO, 99), 189 (25), 174(8.3), 117 (10). HR-MZ m/z
Calcd for C.sub.22H.sub.27N.sub.3O.sub.2: 365.2105; found
365.2100.
[0019]
(-)-(3aS)-1,8-Dibenzyl-3a-methyl-1,2,3,3a,8,8a-hexahydropyrrol[2,3--
b]indol-5-yl N-4'-isopropylphenylcarbamate (compound 45).
(-)-(3aS)-1,8-Dibenzyl-3a-methyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]ind-
ol-5-ol.sup.2 (68 mg, 0.18 mmol) was dissolved in anhydrous ether
(2 mL), and a piece of Na metal (approx. 1 mg) was added. The
mixture was stirred at r.t. for 1 min, then
4-isopropylphenylisocyanate (30 mg, 0.18 mmol) was added and
stirred for 5 min. Evaporation of solvent gave a crude product
[0020] 20 which was directly chromatographed to give 45 (89 ma,
991.3%) as a gum: [.alpha.] D-44.7.degree. (C=0.5, CHCl.sub.3);
.sup.1H NMR (CDCl.sub.3): .delta.7.29 (d, J=8.5 Hz, 2H, C2'-H and
C6'-H), 7.14 (d, J=8.5 Hz, 2H, C3'-H and C5'-H), 6.78 (d, J=2.2 Hz,
1H, C4-H), 6.70(dd, J=2.5, 8.5 Hz, 1H, C6-H), 6.15 (d, J=8.5 Hz,
1H, C7-H), 4.48 (s, 1H, C8a-H), 4.30-4.15 (AB, J=16.6 Hz, 2H,
Ph--CH.sub.2--N8), 3.73 (s, 2H, Ph--CH.sub.2--N1), 2.80 (m, 1H,
--HC<), 2.70 (m, 2H, C2-H.sub.2), 1.90 (m, 2H, C3-H.sub.2), 1.40
(s, 3H, C3a-CH.sub.3), 1.15 (d, J=7.0 Hz, 6H); EI-MS m/z (relative
intensity): 370 (MH.sup.+--ArNHCO--, 1.0), 294 (90), 279 (10), 237
(8.0), 174(95), 160 (92), 132 (60), 104 (55), 91 (100). HR-MZ m/z
Calcd for C.sub.35H.sub.37N.sub.3O.sub.2: 531.2888; found
531.2907.
[0021]
(-)-(3aS)-3a-Methyl-1,2,3,3a,8,8a-hexahydropyrrol[2,3-b]indol-5-yl
N-4'-isopropylphenylcarbamate (compound 41). Compound 45 (42 mg,
0.078 mmol) was dissolved in isopropanol (1 mL) and Pd(OH).sub.2/C
(5 mg) was added. The reaction mixture was stirred under hydrogen
at atmospheric pressure and r.t. for 60 h, then the catalyst was
filtered. Evaporation of solvent gave a residue which was
chromatographed (CH.sub.2Cl.sub.2/MeOH=10/1) to give the most polar
component, compound 41 (14 mg, 51.0%) as a
[0022] 20 gum: [.alpha.]D-71.1.degree. (C=0.3, CHCl.sub.3); .sup.1H
NMR (CDCl.sub.3): .delta.7.29(d, J=8.5 Hz, 2H, C2'-H and C6'-H),
7.10 (d, J=8.5 Hz, 2H, C3'-H and C5'-H), 6.80 (m, 2H, C4-H and
C6-H), 6.55 (d, J=8.5 Hz, C7-H), 5.20 (s, 1H, C8a-H), 2.90 (m, 1H,
Ph--CH<), 2.80 (m, 2H, C2-H.sub.2), 2.13 (m, 2H, C3-H.sub.2),
1.45 (s, 3H, C3a-CH.sub.3), 1.18 (d, J=7.0 Hz, >CMe.sub.2);
EI-MS m/z (relative intensity): 190 (MH.sup.+--ArNHCO, 98),
174(10), 160(70), 146(100), 133(11), 117 (15), 103 (5.0), 91 (14);
HR-MS (NH.sub.3) m/z: Calcd for C.sub.21H.sub.25N.sub.3O.sub.2:
351.1948; found: 351.1941.
[0023]
(-)-3aS)-1,8-Dibenzyl-3a-methyl-1,2,3,3a,8,8a-hexahydropyrrol[2,3-b-
]indol-5-yl N-methylcarbamate [compound
42].(-)-(3aS)-1,8-Dibenzyl-3a-meth-
yl-1,2,3,3a,8,8a-hexahydro-5-methoxypyrrolo[2,3-b]indole (47.5 mg,
0.13 mol) was dissolved in anhydrous ether (2 ml), and a piece of
Na metal (approx. 1 mg) was added. The mixture was stirred at room
temperature for 1 min, then methylisocyanate (14.6 mg, 0.26 mmol)
was added and the mixture stirred for 10 min. Evaporation of
solvent gave a crude product which was directly
[0024] 20 chromatographed to give 42 (50.0 mg, 90.0% as a gum:
[.alpha.]D-58.2.degree. (C=0.7, CHCl.sub.3); .sup.1H NMR
(CDCl.sub.3): .delta.7.40-7.20 (m, 10H, Ar--H), 6.75 (d, J=2.2 Hz,
1H, C4-H), 6.64 (d, J=8.5 Hz, 1H, C6-H), 6.24 (d, J=8.5 Hz, 1H,
C7-H), 4.65 (s, 1H, N--H), 4.40 (s, 1H, C8a-H), 4.35-4.20 (AB,
J=16.6 Hz, 2H, Ph--CH.sub.2--N8), 3.70 (s, 2H, Ph--CH.sub.2--N1),
2.80 (d. J=3.9 Hz, 3H, NH--CH.sub.3), 2.70 (m, 2H, C2-H.sub.2),
1.90 (m, 2H, C3-H.sub.2), 1.35 (s, 3H, C3a-CH.sub.3); EI-MS, m/z
(relative intensity): 370 (MH.sup.+--CH.sub.3NHCO--,33), 354 (1.5),
279 (8.5), 264 (3.0), 91 (100). Anal.
(C.sub.27H.sub.29N.sub.3O.sub.3) C, H, N.
[0025]
(-)-(3aS)-1,8-Dibenzyl-3a-methyl-1,2,3,3a,-8,8a-hexahydropyrrol[2,2-
-b]indol-5-yl N-phenylcarbamate [compound 43].
(-)-(3aS)-1,8-Dibenzyl-3a-m-
ethyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indol-5-ol (40.7 mg. 011
mmol) was dissolved in anhydrous ether (2 ml), and a piece of Na
metal (approx. 1 mg) was added. The mixture was stirred at room
temperature for 1 min, then phenylisocyanate (13.1 mg, 0.11 mol)
was added and stirred for 5 min. Evaporation of solvent gave a
crude produce which was directly
[0026] 20 chromatographed to give 43 (48 mg, 89.1%) as a gum:
[.alpha.D-59.1.degree. (C=0.7, CHCl.sub.3); .sup.1H NMR
(CDCl.sub.3): .delta.7.40-6.90 (m, 15H, Ar--H), 6.75 (d, J=2.5 Hz,
1H, C4-H), 6.73 (d, J=8.5 Hz, 1H, C6-H)m 6.17 (d, J=8.5 Hz, 1H,
C7-H), 4.45 (s, 1H, C8a-H), 4.30-4.20 (AB, J=16.6 Hz, 2H,
Ph--CH.sub.2--N8), 3.72 (s, 2H, Ph--CH.sub.2N1), 2.70 (m, 2H,
C2-H.sub.2), 1.90 (m 2H, C3-H.sub.2), 1.38 (s, 3H, C3a-CH.sub.3);
EI-MS, m/z (relative intensity): 370 (MH.sup.+--PhNHCO--, 31), 354
(1.0), 279 (8.0), 264 (2.0), 91 (100). Anal.
(C.sub.27H.sub.29N.sub.3O.sub.3) C, H, N.
[0027] The synthesis of the remaining compounds listed in Table 1
are known, and may be found in the references cited for each
compound listed in FIG. 1. Each of the publications are hereby
incorporated by reference into the disclosure hereof.
[0028] In summary of Table 1, extensive studies have demonstrated
that whereas the classical anticholinesterase physostigmine (1)
possesses no selectivity of inhibitory action between the two
enzyme subtypes, acetyl-(AChE) and butyrylcholinesterase (BChE),
specific substitutions in the 4' (para) position, (4,5), of the
(-)-phenylcarbamate of physostigmine (2) provide compounds with a
selectivity for BChE inhibition. This is unexpected as other 4'
substitutions, (6), or similar substitution in other positions,
such as at the 2' (ortho) position (3,7), provide no selectivity
for BChE. Recent studies have shown that 3' substitution likewise
does not provide BChE selectivity, for example 3'-methyl-carbamoyl
eseroline has an IC.sub.50 of AChE 27 nM and BChE 165 nM. Indeed,
substitution in the 2'-; 2', 4'-; 3'-; 2', 3'-; 3', 5'-; or 2', 4',
6'-positions do not provide BChE selectivity. Additional studies
have demonstrated that, independently, substitutions in the;
N.sup.1-position of physostigmine (1), such as
N.sup.1-norphysostigmine (8), N.sup.1-benzylnorphysostigmine (12),
N.sup.1-phenethylnorphysostigmi- ne (16), and
N.sup.1-allylnorphysostigmine (20) provide BChE selectivity,
compared to physostigmine (1). This is unexpected as other
substitutions, such as amines (21), do not. Replacement of the
N.sup.1 group of physostigmine (1) to provide thiaphysovenine (22)
and physovenine (26) also unexpectedly provides a selectivity of
inhibitory action for BChE. Yet further studies have demonstrated
that, independently, substitution in the N.sup.8-position of
physostigmine (1), to provide N.sup.8-benzylnorphysostigmine (30)
and N.sup.8-norphysostigmine (34), produce potent and selective
inhibitors of BChE. A combination of the described modifications
provides compounds [11, 15, 6, 19, 25, 29, 33, 37] which exhibit or
are expected to exhibit dramatic selectivity for BChE versus AChE
inhibitory action. Other useful compounds for the purpose of this
invention include compounds [12, 20 and 22].
[0029] A particularly preferred compound for use in the method of
the present invention is. cymserine (Compound 4, Table 1, FIG. 1A).
The preference for cymserine is based on its ease of synthesis, the
availability of stable salts, and its ability to cross the blood
brain barrier.
[0030] Compositions for use in the methods of the invention include
compositions wherein the active ingredient is contained in an
effective amount to achieve its intended purpose. The compounds can
be administered in any pharmaceutically acceptable amount, for
example, in amounts ranging from 0.001 gram to about 1 gram per
kilogram of body weight. Based on the information which is
presented herein, the determination of effective amounts is well
within the skill of the ordinary practitioner in the art. The
compounds are generally used in pharmaceutical compositions (wt %)
containing the active ingredient with a carrier or vehicle in the
composition in an amount of about 0.1 to 99 wt % and preferably
about 25-85 wt %.
[0031] Either fluid or solid unit dosage forms can be readily
prepared for oral administration. For example, the highly selective
BChE inhibitors can be admixed with conventional ingredients such
as dicalcium phosphate, magnesium aluminum silicate, magnesium
stearate, calcium sulfate, starch, talc, lactose, acacia, methyl
cellulose and functionally similar materials as pharmaceutical
excipients or carriers. A sustained release formulation may
optionally be used. In older or incoherent patients sustained
release formulations may even be preferred. Capsules may be
formulated by mixing the compound with a pharmaceutical diluent
which is inert and inserting this mixture into a hard gelatin
capsule having the appropriate size. If soft capsules are desired,
a slurry of the compound with an acceptable vegetable, light
petroleum or other inert oil can be encapsulated by forming into a
gelatin capsule.
[0032] Suspensions, syrups and elixirs may be used for oral
administration or fluid unit dosage forms. A fluid preparation
including oil may be used for oil soluble forms. A vegetable oil
such as corn oil, peanut oil or a flower oil, for example, together
with flavoring agents, sweeteners and any preservatives produces an
acceptable fluid preparation. A surfactant may be added to water to
form a syrup for fluid unit dosages. Hydro-alcoholic pharmaceutical
preparations may be used having an acceptable sweetener, such as
sugar, saccharin or a biological sweetener and a flavoring agent in
the form of an elixir.
[0033] Pharmaceutical compositions for parenteral and suppository
administration can also be obtained using techniques standard in
the art.
[0034] Preferred uses of the compounds according to the invention
are as pharmaceutical agents suitable for oral administration.
Another preferred use of the compounds is in transdermal parenteral
formulations, which are particularly useful in preventing or
treating cholinergic disorders such as Alzheimer's disease.
Accordingly, compositions suitable for administration to these
areas are particularly included within the invention. The above
parenteral solutions or suspensions may be administered
transdermally and delivered with a skin patch. If desired they may
be given by injection in an appropriate vehicle such as sesame
oil.
[0035] Accordingly, incorporation of the active compounds and a
slow release matrix may be implemented for administering
transdermally. The compounds may be administered transdermally in
amounts of about 0.01 to 99% of the composition and preferably
about 25 to 85 wt % of the active ingredient in the vehicle or
carrier.
[0036] Transdermal therapeutic systems are self-contained dosage
forms that, when applied to intact skin, deliver drug(s) at a
controlled rate to the systemic circulation. Advantages of using
the transdermal routing include: enhanced therapeutic efficacy,
reduction in the frequency of dosing, reduction of side effects due
to optimization of blood concentration vs. time profile, increased
patient compliance due to elimination of multiple dosing schedules,
bypassing the hepatic "first pass" metabolism, avoiding
gastrointestinal incompatibilities and providing a predictable and
extendible duration of activity. However, the main function of the
skin is to act as a barrier to entering compounds. As a
consequence, transdermal therapy has been preferred for a limited
number of drugs that possess the desirable physiochemical
properties for diffusion across the skin barrier. One effective
method of overcoming the barrier function of the skin is to include
a penetration enhancer in the formulation of the transdermal
therapeutic system.
[0037] The penetration enhancer is a chemical compound that, when
included in a formulation, temporarily increases the permeability
of the skin to a drug line allowing more of the drug to be absorbed
in a shorter period of time. Several different types of penetration
enhancers have been reported such as dimethylsulfoxide,
n-decylmethylsulfoxide, N,N-dimethylacetamide,
N,N-dimethylformamide, 1-dodecylazacycloheptane-2-one (Azone),
propylene glycol, ethanol, pyrrolidones such as
N-methyl-2-pyrrolidone (NMP) and surfactants.
[0038] The above compounds can be present in the reservoir alone or
in combination with pharmaceutical carriers. The pharmaceutical
carriers acceptable for the purposes of this invention are the
known art carriers that do not adversely effect the drug, the host,
or the material comprising the drug delivery device. Suitable
pharmaceutical carriers include sterile water, saline, dextrose,
dextrose in water or saline, condensation products of castor oil
and ethylene oxide combining about 30 to 35 moles of ethylene oxide
per mole of castor oil, liquid acid, lower alkanols, oils such as
corn oil, peanut oil, sesame oil and the like, with emulsifiers
such as mono- or di-glyceride of a fatty acid; or a phosphatide,
e.g., lecithin, and the like; glycols, polyalkylene glycols,
aqueous media in the presence of a suspending agent, for example,
sodium carboxymethyl cellulose, sodium alginate,
poly(vinylpyrrolidone), and the like, alone or with suitable
dispensing agents such as lecithin, polyoxyethylene stearate, and
the like. The carrier may also contain adjutants such as preserving
agents, stabilizing agents, wetting agents, emulsifying agents and
the like together with penetration enhancer and the compounds of
this invention.
[0039] The effective dose for mammals may vary due to such factors
as age, weight, activity level or condition of the subject being
treated. Typically, an effective dosage of a compound according to
the present invention is about 1 to 800 milligrams when
administrated by either oral or rectal dose from 1 to 3 times
daily. This is about 0.002 to about 50 milligrams per kilogram of
the subject's weight administered per day. Preferably about 10 to
about 300 milligrams are administered orally or rectally 1 to 3
times a day for an adult human. The required dose is considerably
less when administered parenterally. Preferably about 0.01 to about
150 milligrams may be administered intramuscularly or
transdermally, one or two times a day for an adult human.
[0040] Compounds for use in the present invention may be
administered topically in amounts of about 0.01 to about 99 wt % of
the composition, and preferably about 25 to 85 wt %. The method
according to the invention comprises administering an effective
amount of a compound according to the invention or an effective
amount of a pharmaceutical composition according to the invention
to a mammal in need of such treatment.
[0041] In the present study we assessed the effects of chronic
cymserine treatment (5 days) on performance of aged Fischer-344
(F344) rats (21-22 months old) in a 14-unint T-maze, referre to as
the Stone maze. The use of the Stone maze paragigm for this study
can be supported by ntwo previous observations: (1) the known
involvement of the cholinergic system in performance as
demonstrated by pharmacological and lesion studied and (2) the
marked age-related decline in performance demonstrated in several
rodent strains including the Fischer 344 (F344) strain. The use of
the F344 rat for this study is also justified because of documented
age-related decline in cholinergic markers in specific brain
regions and the demonstrated improvement in memory performance of
aged rats from this strain following various cholingeric
treatments.
[0042] Male F344 rats 21-22 months old were obtained from
Harlan-Sprague-Dawley under contract from the National Institute on
Aging. They were maintained two per cage in a vivarium at the
Gerontology Research Center under specific pathogen free conditions
as characterized previously.
[0043] As previously described, the Stone maze is constructed of
translucent plastic with a grid floor wired for scrambled foot
shocks and is surrounded by gray walls to reduce availability of
extra-maze cues. The only other apparatus was a straight runway (2
m) used for pretraining. Similar to the maze, the runway was also
constructed of translucent plastic, and contained a grid floor
wired for scrambled foot shocks, and was surrounded by gray
walls.
[0044] Beginning on day 1 rats received a single daily i.p.
injection of either 0.9% NaCl as the saline control group or
cymserine tartrate dissolved in saline and given in does of 0.5 and
1.0 mg/kg that continued on days 2-5. On days 3-5 injections were
made 30 min prior to behavioral testing.
[0045] Beginning on day 2 rats were provided training in one-way
active avoidance in the straight runway. On each trial, the rat had
to locomote from the start box to the goal box within 10 s to avoid
the onset of foot shock (0.8 mA). Rats received 10 trials on day 2
and 10 trials on day 3. Training was terminated when the rat met a
performance criteria of eight avoidances within 10 consecutive
trials within a maximum of 30 trials. Only rats meeting this
criterion were tested in the Stone maze on the next day.
[0046] Rats received training in the Stone maze scheduled as a
4-trial session during the morning and afternoon on days 4 and 5.
During each trial, the rat had to locomote from a start box to a
goal box through five maze segments each separated by guiiltine
doors. The reinforcement contingency required the rat to negotiate
each segment within 10 s to avoid the onset of mild foot shock (0.8
mA), which was terminated when the animal moved through the door
into the next segment. After entry into succeeding segments, the
door from the preceding segment was closed to prevent backtracking.
Recorded as deviations from the correct pathway, errors were the
primary dependent variable and were counted automatically by a
series of infrared sensors connected to a microprocessor. Run time
from the start box to the goal was also recorded automatically. The
frequency and duration of foot shock were recorded on a
mechanically operated clock.
[0047] No effects of drug treatment were observed during
pretraining. The mean (s.e.m.) avoidances for all rats were 67%
(1.7), 65% (2.8), 63% (3.8), and 61% (3.9) for the control, and 1,
2 and 3 mg kg cymserine groups, respectively. A one-way analysis of
variance (ANOVA) revealed no significant group difference in this
parameter, F(3,40)<1.0.
[0048] Cymserine treatment significantly reduced the number of
errors made in the Stone maze compared with the control condition
(FIG. 5). This effect was most prominent during the last blocks of
training and appeared least effective for the 3 mg kg dose.
Statistical confirmation was provided in the results of a four
(drug group) by four (trial block) ANOVA with repeated measures on
the last factor. The results yielded a significant main effect of
group, F(3,42)=3.41, p=0.03, a significant main effect of trial
block, F(3,126)=151.6,p<0.0001, but the group by block
interaction did not reach statistical significance F(9,126)=1.55,
p=0.13. Thus, individual comparisons of errors were made across all
trials. Only the 1 and 2 mg kg groups exhibited significantly
improved performance compared with controls.
[0049] Other performance variables (runtime, shock frequency and
duration) were also reduced in cymersine treated rats to a less
consistent degree than observed for errors. The data for each
variable were first analyzed in a four (drug) by four (blocks)
ANOVA with repeated measures on the last block. No significant main
effect of drugs emerged from these analyses; however, the drug x
block interaction was significant (p<0.005) in each. Thus, the
data were analyzed further using t-test comparisons to the control
groups at each block. Rats treated with 1.0 mg kg cymserine
exhibited significantly reduced run time, shock frequency and
duration at blocks 3 and 4 compared with controls. In the 2.0 mg kg
group these performance parameters were significantly reduced at
block 4. The 3.0 mg kg group was significantly different from
controls only for shock duration at block 4. Shock duration was
also significantly reduced at block 2 in the 1.0 mg kg group. In
summary, these perfomance variables were significantly affected
only during the later trials and most prominently in the 1.0 mg kg
group. Some motoric side effects such as fine tremor were detected
in a few rats treated with the 3 mg kg dose; otherwise, no side
effects were noted in cymserine treated rats.
[0050] Chronic treatment with cymserine markedly improved the
learning performance of aged rats in the Stone maze. Rats receiving
doses of 1-2 mg kg cymserine exhibited signifcantly reduced errors
as well as improvement in other performance variable during the
last few trials. This response was presumably due to enhanced
cholinergic neutrotransmission by the action of this potent,
long-acting cholinesterase inhibitor.
[0051] A further aspect of the present invention relates to the
discovery that highly selective BChE inhibitors may be used in
reducing beta-amyloid precursor protein synthesis and secretion.
Alzheimer's disease is characterized by depositions of the amyloid
beta-peptide (A.beta.) in the form of cerebrovascular amyloid and
extracellular senile plaques. The primary core constituent of
senile plaques is A.beta. peptide, a self-aggregating protein of 39
to 43 residues, which is derived from a group of larger
glycosylated transmembrane proteins, .beta.-APPs, of 695 to 770
amino acids. .beta.-APP is the source of the toxic A.beta.
peptides, known to deposit in the brain of AD patients. Mutations
of the APP gene cosegregate with AD in certain families implicating
.beta.-APP695 to .beta.-APP770, some of which contain the active
Kunitz family of serine protease inhibitor (KPI) domain, as well as
A.beta. and other amyloidogenic fragments of .beta.-APP as central
in the disease process. (Selkoe, J. Neuropathol., Exp. Neurol.
53:438-447, 1994) .beta.-APP is processed/metabolized by
alternative proteolytic pathways to generate different breakdown
products. These include a secretory and a lysosomal/endosomal
pathway. In the secretory pathway, three different secretases have
been implicated. In man, but not rat, the majority of .beta.-APP is
cleaved within the A.beta. region by .alpha.-secretase to generate
non-amyloidogenic soluble .beta.-APP, sAPP, which is known to
possess a number of valued physiological roles. A postulated
alternative secretase cleavage, .gamma.-secretase, generates a
truncated sAPP.gamma. which contains a potentially amyloidogenic
sequence. This is the preferential form produced in the rat, and
further cleavage in the human, by a postulated .beta.-secretase,
produces the neurotoxin A.beta.. (Checler, J. Neurochem.,
65:1431-1444, 1995.)
[0052] Synthesis, processing and secretion of .beta.-APP and its
derivatives occur in vivo in brain, with the products being
detectable in brain and CSF in both man and animal models, and,
additionally, occurs in vitro in tissue culture with the products
being detectable in the conditioned medium of cell cultures and in
the cell lysates. Factors that regulate depositions of A.beta. are
central to understanding the cerebrovascular changes in AD.
(Roberson & Harrell, Brain Res. Rev., 25:50-69, 1997.) This
disease is also marked by the dramatic loss of cholinergic neurons
that project to the cortex and neurochemically by a reduction in
presynaptic (choline acetyl transferase, ChAT) markers of the
cholinergic system, particularly in the areas of the brain related
to memory and learning. (Perry, et al., 1978, ibid.) There is a
clear relationship between the loss of cholinergic projections to
the cortex and hippocampus and the synthesis and processing of
.beta.-APP. (Wallace, et al., PNAS, 98:8712-8716, 1993.) The
cholinergic deficits of AD have been modeled in the rat by
neurotoxic lesions in the basal forebrain, the most common one
being of the nucleus basalis of Meynert (nbM) (Olton and Wenk, In,
Psychopharmacology: The Third Generation of Progress (ed, Meltzer)
Raven Press, NY, pp 941-954, 1987). Such lesions in rat, like AD in
man, lead to a depleted cholinergic system in the cortex of animals
and cognitive impairments with features common to AD (Kesner et
al., Behav. Neurosci., 101:451-456, 1987). Furthermore, cholinergic
forebrain lesions significantly increase the levels of .beta.-APP
mRNA in cortex and secreted .beta.-APP, containing the fill length
of A.beta., in the CSF of rats (Wallace et al., Mol. Brain Res. 10:
173-178, 1991).
[0053] In a report by Lahiri et al., (ANN.NY. Acad. Science,
828:416-421, 1997.) the possibility that the processing of
.beta.APP can be regulated by different cholinesterase inhibitors
was investigated. The drugs that were studied in determining the
effects of cholinesterase inhibitors on the secretion of secreted
forms of .beta.APP from a number of cell lines, i.e., glioblastoma,
HeLa, neuroblastoma and PCI2, included 3,4 diaminopyridine,
metrifonate, physostigmine (compound 1, Table 1) and tacrine. The
results observed led to the statement that treating neuronal cells
with tacrine did regulate the secretion of .beta.APP, reducing it,
while none of the other drugs, all classical anticholinesterases,
produced a change in such secretion. It was suggested that any
activity shown by these other agents may be independent of their
anticholinesterase activity. None of the compounds utilized in
these studies was a highly selective BChE inhibitor.
[0054] In further studies, the actions of selective AChE (Compounds
2, 3, (phenserine and tolserine)Table 1) and BChE (Compound 4
(cymserine), Table 1) inhibitors were assessed against human
neuroblastoma cell lines. Both secreted levels, measured in the
conditioned medium, and cellular levels of .beta.APP, measured in
the cell lysates, were assessed and compared to levels achieved in
untreated cells. Cymserine dramatically reduced cellular levels of
.beta.APP, indicating a reduced synthesis, and, likewise, reduced
secreted .beta.APP levels (FIG. 2).
[0055] A methodology that can be employed to determine the ability
of a selected compound to regulate cellular and secreted .beta.APP
levels is as follows:
[0056] 1 to 1.5.times.10.sup.7 of each cell type (neuronal and
non-neuronal) are cultured in their respective medium. Before
adding drug, the cells are fed with media containing only 0.5% of
FBS (low serum). The cells are then incubated either in the absence
or presence of the test compound. Following incubation periods from
12 to 48 hours, the conditioned medium from each plate is collected
and both it and the cells are centrifuged at 800 g. for 10 minutes.
The conditioned medium is collected and the cells lysed in buffer
containing 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 2 mM
PMSF, 0.5% sodium deoxycholate, 1 ug/ml each of aprotonin,
leupeptin and TLCK, and 0.1 ug/ml of pepstatin A. The cells are
centrifuged for 10 min at 11,000 g at 4.degree. C. Proteins of the
supernatant solution (cell lysate) are measured by the Bradford
dye-binding procedure (Bradford, Anal. Biochem. 72: 248-254,
1976).
[0057] For polyacrylamide gel electrophoresis and immunoblotting:
in control experiments, an equivalent concentration of ethanol was
used as a vehicle which is less than 1% in media. Compound dosages
of from about 0.15 mM to 0.5 mM may be used. 100 uL of conditioned
media or 30 ug of protein from the total cell lysate is separated
on a 12% polyacrylamide gel containing SDS (SDS-PAGE). Immunoblot
analysis was performed using the avidinbiotyinylated complex
detection kit of Vector Laboratories as described by Lahiri, et
al., J. Neurosci: Res. 12:777-787 (1994). The antisera employed is
from the mAb22C11 clone (Boehringer Mannheim) which recognizes all
mature forms of .beta.APP found in cell membranes as well as the
carboxyltruncated soluble forms secreted into the conditioned media
and the APP-like protein. Additionally, the mAb6E10 is employed
which recognizes residues 1 to 28 of A.beta.. To ensure that drug
effects are selective to .beta.APP and do not unselectively affect
all proteins, antibody raised against human protease nexin II
(PN-II) and anti-HSP-70 (an antibody raised against heat shock
protein-70, HSP-70) can be used. Biotinylated secondary antibodies,
horse anti-mouse and goat anti-rabbit (Boehringer Mannheim, and
Vector Labs) are also used. The above assay method is used to
identify which of the highly selective BChE inhibitors of the
present invention demonstrate the ability to regulate the synthesis
and secretion of secreted and cellular forms of .beta.APP. As shown
in FIG. 2, cymserine can dramatically reduce .beta.APP levels, and
does so when assessed by both mAb 22C11 and 6E10 to .beta.APP
without affecting other secretory proteins, such as HSP-70.
[0058] Cymserine, a representative of selective BChE inhibitors,
additionally alters .beta.APP synthesis/processing in vivo. In rats
with lesions of the cholinergic forebrain (nucleus basalis of
Meynert), levels of .beta.APP are immediately and dramatically
increased in the CSF (FIG. 3, control group with lesion vs. control
sham), as a consequence of a depleted presynaptic cholinergic
system, modeling AD, and reduced cholinergic projections to higher
brain centers in the cortex and hippocampus, as shown previously
(Wallace, et al., 1993 & 1995 ibid). Studies have demonstrated
that, unlike man, secreted .beta.APP contains the full length of
A.beta., but it is not cleaved by secretase action in rat to
produce toxic A.beta. peptide (Wallace et al., J. Neurosci.,
15:4896-4905, 1995), and hence AD is unique to man, Therefore, in
rat, the elevation of .beta.APP that contains the full length of
A.beta. peptide, models increased production of A.beta. in man.
[0059] The administration of cymserine to rats with forebrain
cholinergic lesions blocks the elevation of secreted .beta.APP
(FIG. 3). This is in accord with the in vitro action of cymserine
on .beta.APP, and additionally demonstrates that systemically
administered cymserine given by the intraperitoneal route twice
daily for 7 days, can readily cross the blood-brain barrier and
enter brain. Indeed, as shown in FIG. 4, cymserine readily enters
and is maintained in brain at levels some 40-fold higher than those
in plasma, following its systemic administration (1 mg/kg by the
intravenous route). Additionally, as shown in FIG. 3, cymserine
reduced levels of .beta. APP in animals without cholinergic
lesions, i.e., in cymserine alone versus pure control rats.
[0060] A methodology that can be employed to determine the ability
of a selected compound to regulate secreted .beta.APP levels in
vivo is as follows:
[0061] For forebrain cholinergic system lesions, rats receive a
unilateral subcortical lesion of the nucleus basalis of Meynert
(nbM) using N-methyl-D-aspartate (NMDA) as an excitotoxin. In
undertaking this, rats are anesthetized and placed in a stereotaxic
apparatus with the upper incisor bar set level with the intra-aural
line. A 33 gauge infusion cannula is lowered into two sites within
the nbM on one side of the brain (AP bregma, ML -2.8 mm, DV -8.0 mm
re: skull and AP bregma -0.8 mm, ML -3.0 mm, DV -7.8 mm,
respectively). One microliter of a solution of 50 mM NMDA in PBS
buffer (physiological pH) is slowly infused into each site.
Controls for the lesion are vehicle alone and the contralateral
side of NMDA treated animals.
[0062] Collection of CSF for analysis of .beta.APP levels is
undertaken by withdrawal of an aliquot from the cistern magna of
rats immediately following their death. Quantitation of .beta.APP
is undertaken by immunoblot analysis utilizing mAb 22C11.
[0063] Determination of the brain and plasma time-dependent
kinetics of cymserine in the rats is undertaken by administering
compound into the saphenous vein of anesthetized animals. Animals
are killed by excess anesthetic at specific times and blood and
brain samples are immediately taken. The blood is centrifuged
(10,000 g, 2 min), plasma is collected and, together with the
sample of brain, is stored at -80.degree. C. Quantitation of
concentrations of cymserine is undertaken by high performance
liquid chromatography.
[0064] In a further embodiment of the present invention the highly
selective BChE inhibitors can be modified by introduction of a
fluorescent label and the resulting labeled reagent can be used in
histochemical detection of lesions or pathologic states associated
with Alzheimer's disease and other dementias on sectioned brain
tissue. Any suitable fluorescent label known in the art can be
employed, such as, for example, fluorescein. The labeled derivative
of the highly selective BChE inhibitor can be prepared in a manner
known per se, such as, for example, by reacting such inhibitor with
5-[4,6-dichlortriazen-2-yl-amino] fluorescein and the reaction
product can be purified by methods known in the art, such as, for
example, thin layer chromatography.
[0065] Derivatives of the highly selective BChE inhibitors which
can be employed in carrying out brain scans, particularly positron
emission tomography and single photon emission tomography, include
the following: compounds in Table 1, and analogues thereof, with
appropriate moieties to provide them either fluorescence or
detection for in vitro or in vivo imaging/quantitation.
[0066] Thus the following radiopharmaceutical agents with
appropriate modification, if needed, can be attached using methods
well known in the art to any of the highly selective BChE
inhibitors: thallium, technetium, iodine.sup.131 or iodine.sup.123,
xenon.sup.133, krypton.sup.481, gallium.sup.67, indium.sup.111,
carbon.sup.11, nitrogen.sup.13 and fluorine.sup.18. These isotopes
can be introduced, for example, as cold kits and are reconstituted
with the appropriate chemicals. The reconstituted compounds, after
administration to the patient, are distributed within the body
according to the physical and chemical properties of the specific
agents as well as the moeity to which the radioactive label is
attached.
[0067] These agents can be used for diagnosis in the brain as well
as the systemic peripheral areas of the body; example include
deposition of peripheral amyloid in the spleen, as well as in the
brain in Alzheimer's disease. Attaching these radioactive agents,
using methods well known in the art, to carrier molecules that pass
the blood brain barrier, e.g.cymserine, can create specific
neuropathology diagnostic agents.
[0068] An example is the use of technetium pertechnate used in
brain imaging. This agent, combined with the highly selective
butyrylcholinesterase inhibitors of the present invention will
localize in certain sections of the brains such as the choroid
plexus.
[0069] The optimal type of radiopharmaceutical has most of its
energy in the form of gamma rays. The diagnostic equipment used in
nuclear medicine has certain optimum detection energy levels. Most
of the radioactive materials used for nuclear medicine are made by
converting stable elements into radioactive forms. The conversion
is performed by nuclear reactors or cyclotrons which bombard the
stable elements with protons or neutron.
[0070] Advances in filmless detectors provide information abut the
number of photons impinging on a sensitive element. This data,
combined with the use of data processing algorithms have increased
the power of medical imaging. The diagnostic imaging devices
include computed tomography (CT), positron emission tomography
(PET) Single Photon Emission Computed tomography (SPC), Digital
Subtraction Angiography (DSA), Angiographic Imaging with synchotron
radiation and Magnetic Resonance Imaging (MRI).
[0071] The principle isotopes used in imaging are carbon.sup.11,
oxygen.sup.15, nitrogen.sup.13. These agents are neutron poor,
positron emitting isotopes. They are produced by a cyclotron and
rapidly incorporated into the compounds of the present
invention.
[0072] Thus, an embodiment of this aspect of the invention are
compounds exhibiting highly selective butyrylcholinesterase
inhibiting activity which are prepared incorporating a
radiopharmaceutical agent thus providing an agent suitable for
patient imaging for the detection for the presence of lesions or
pathological states associated with Alzheimer's disease. Such
agents can be introduced by sympathetic pathways known in the art.
Thus, for example, the carbomyl moeity of the highly selective
butyrylcholinesterase inhibitors can be provided as an .alpha.
carbon.sup.11 moeity by using the general procedures used by
Bonnot, J. Label Comp. Radiopharm. 33 [4}, 277-284 (1993). The
resulting compounds can be administered parenterally to the patient
and will pass to the brain where they will selectively bind to any
lesions or pathological states and can be detected by suitable
imaging equipment such as a PET scan.
[0073] References
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